Project Summary Immunization of rhesus cytomegalovirus (RhCMV)-positive rhesus macaques (RM) with the prototype RhCMV68-1-vectored simian immunodeficiency virus (SIV) vaccine expressing Gag, Retanef, Pol and Env is effective in approximately 50% of animals, with protected animals demonstrating almost complete control of systemic SIV infection following mucosal challenge with pathogenic SIV. However, efficacy occurs in the absence of vaccine-induced antibody (Ab) responses, and is instead associated with unconventional anti-SIV MHC-II and MHC-E-restricted CD8 T cell responses. Although this level of SIV control is remarkable, further development of the CMV vaccine platform will clearly be necessary to induce a humoral response to the SIV Env protein: a response considered by many to be essential for an efficacious vaccine. The conditions necessary for induction of such desired Ab responses are currently unclear. Our preliminary data indicate that RhCMV expressing SIV Gag is capable of inducing Gag-specific Ab, but only in RhCMV-negative RM. The same RhCMV-Gag vaccine does not induce comparable Ab responses in RhCMV-positive RM. This finding is consistent with previous published reports showing either negligible or no induction of SIV Env-specific Ab in RhCMV-positive RM vaccinated with RhCMV-Env. These findings suggest that pre-existing RhCMV immunity may be one critical factor that restricts Ab induction to vaccine antigens delivered by RhCMV vectors using protocols based on RhCMV SIV vaccines alone. Magnitude of vaccine antigen expression from prototype RhCMV vaccines may be another factor, particularly for the RhCMV-Env vaccine that was intentionally designed for low expression of the ?toxic? Env gene. We propose that robust antigen expression will also be critical for Ab induction in RhCMV-positive RM immunized with RhCMV-SIV vaccines. The current proposal is an exploratory investigation into i) approaches to promote humoral responses to RhCMV-vectored SIV vaccines in the face of pre-existing RhCMV immunity, and ii) strategies to stably increase SIV antigen expression. This investigation is based on our overall hypothesis that pre-existing RhCMV immunity and magnitude of SIV antigen expression are critical modifiable factors contributing to restricted humoral responses observed in RhCMV-positive macaques. Aim 1 will investigate induction of SIV-specific Ab in RhCMV-positive macaques by two distinct prime-boost protocols that utilize recombinant subunit proteins (Env and Gag) and RhCMV-SIV vaccines. Immunized animals will be examined for circulating and mucosal Ab responses, B cell and T cell responses in blood and lymph nodes. Aim 2 focuses on the generation of candidate RhCMV human immunodeficiency virus (HIV)-1 Env vaccines designed to enhance Env expression using a novel strategy recently described for a modified RhCMV-vectored Ebola virus vaccine. Proposed studies will provide a timely foundation for future investigation into combination RhCMV-SHIV vaccine protocols that include recombinant Env proteins together with RhCMV-Env vaccines designed for improved CMV-HIV vaccine protective efficacy.